The major goal of the proposed research is to elucidate mechanisms that underlie transport of membranous organelles that are related to plasma membrane renewal (i.e., recycling) in axons. The central hypotheses that serve as rationales for the project are that a spectrin-actin and/or a myosin-actin cytoskeletal system(s) is/are involved in the 'packaging' and/or transport of cytomembranes targeted for recycling of the axolemma. The research will focus on two model systems: (1) ganglion cell axons (RGC axons) regenerating in culture from goldfish retinal explants; and (2) the large myelinated axons of the pair of Mauthner neurons in goldfish that extend the full length of the neuroaxis below the pons. Large varicosities and small intervening phase-dense inclusions are visible motile structures associated with regenerating RGC axons. These structures contain a 'packaged' aggregate of cytomembranes which are destined for insertion at the growth cone during elongation, or for the cell body after retrieval. Actin, spectrin, putative myosin and calmodulin are concentrated in these motile structures, and are co-transported with the contents of the varicosities and inclusions. In Mauthner axons, a spectrin-actin cytoskeletal system is localized near the axon's surface, as are also putative myosin and calmodulin, presumably within the subaxolemmal domain where hypolemmal cisternae are also located. Lectin binding studies suggest that membranes of hypolemmal cisternae contain glycoconjugates, similar to plasma membrane. Thus, the subaxolemmal space may represent a special transport pathway for precursor plasmalemma. The major objectives are: (i) to investigate the potential role of the spectrin-actin network in packaging/transport of cytomembranes in varicosities and intervening phase-dense inclusions in RGC axons and of hypolemmal cysternae in the M-axon; (ii) to investigate mechanisms whereby calcium/calmodulin may regulate cytoskeletal organization and control movement; (iii) to investigate the potential role of a myosin-actin system in the packaging/transport of cytomembranes in RGC axons and the M-axon; and (iv) to utilize in vitro and in situ Mauthner neuron preparations for studying rapid transport in the axon.